HISTOLOGY AND TISSUE STAINING Modern Techniques in Life Sciences 09.05.2017, Sandra Ritz Microscopy & Histology & Staining Greek: ἱστός histos „tissue“ und ‐logy, gr. λόγος logos „study of“ Marcello Malpighi (1628‐1694): Malpighian tubules, Malpighian corpuscles…) Antonie van Leeuwenhoek (1632‐1723): magnifying lens, dyes Lens Sample holder Focus croccus ‐ Single cell organisms “animalculus” ‐ Sperms, blood cells, bacteria ‐ Banded pattern of muscular fibers ‐ Stains samples with saffron www.imb‐mainz.de Jeroen Rouwkema - http://www.flickr.com/photos/rouwkema/2262158965/ Microscopy Core Facility Microscopy & Histology & Staining Marcello Malpighi (1628‐1694): Malpighian tubules, Malpighian corpuscles… Antonie van Leeuwenhoek (1632‐1723): magnifying lens, dyes Robert Hooke (1635‐1703): Micrographia (1665) 1832: Matthias Schleiden and Theodor Schwann: Cell theory „All plant and animal tissues are composed of cells.“ www.imb‐mainz.de https://ceb.nlm.nih.gov/proj/ttp/books.htm Microscopy Core Facility Microscopy & Histology & Staining Antonie van Leeuwenhoek (1632‐1723), (microscopy, dyes) Marcello Malpighi (1628‐1694): Malpighian tubules, Malpighian corpuscles… Robert Hooke (1635‐1703): Micrographia 1832: Matthias Schleiden and Theodor Schwann: Cell theory „All plant and animal tissues are composed of cells.“ Joseph von Gerlach (1820‐1896): carmine/gelatin mixture for staining natural stainings: saffron, carmine/cochineal (E120), hematoxylin Cochineal (Dactylopius coccus) www.imb‐mainz.de https://de.wikipedia.org/wiki/Cochenilleschildlaus#/media/File:Indian_collecting_cochineal.jpg Microscopy Core Facility Microscopy & Histology & Staining Antonie van Leeuwenhoek (1632‐1723), (microscopy, dyes) Marcello Malpighi (1628‐1694): Malpighian tubules, Malpighian corpuscles… Aniline Robert Hooke (1635‐1703): Micrographia 1832: Matthias Schleiden and Theodor Schwann: Cell theory „All plant and animal tissues are composed of cells.“ Joseph von Gerlach (1820‐1896): carmine/gelatin mixture for staining natural stainings: saffron, carmine/cochineal 19th century: WH Perkin synthesized the first synthetic „Mauveine“ aniline dye „aniline purple or Mauveine“ (Malve) or aniline purple extracted from coal tar (Steinkohlenteer). www.imb‐mainz.de https://de.wikipedia.org/wiki/Cochenilleschildlaus#/media/File:Indian_collecting_cochineal.jpg Microscopy Core Facility Histology/Histopathology today Depends entirely on the experiment. E.g.: HE Which molecules are you interested in? What kind of tissue are you using? Do you want to detect active enzymes? Does your AB work in fixed tissue? IF IHC ISH/FISH EM A BC D HE: Pankreas by user:Polarlys ‐ Eigenes Werk, CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=1032935 A: Porcine blood vessels by Alizee Pathology LLC. ‐ http://alizeepathology.com/samples/1, CC BY‐SA 3.0, https://commons.wikimedia.org/w/index.php?curid=33313176 B: Kidney IHC agianst CD10, by Nephron ‐ Own work, CC BY‐SA 3.0, https://commons.wikimedia.org/w/index.php?curid=5961392 C: ISH and FISH, by Ryan Jeffs ‐ Own work, CC BY‐SA 3.0, https://commons.wikimedia.org/w/index.php?curid=21438920 D: Immunogold for mtDNA, by Francisco J Iborra1 , Hiroshi Kimura2 and Peter R Cook ‐ The functional organization of mitochondrial genomes in human cells, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=9085139 www.imb‐mainz.de Microscopy Core Facility Histology Tissue (from organ preparation/biopsy) Fixation/snap freezing/fixing and freezing Sectioning Staining Mounting Microscopy www.imb‐mainz.de https://www.sciencedump.com/sites/default/files/styles/article_width/public/field/image/Schermafbeelding%202015‐ Microscopy Core Facility 12‐16%20om%2021.28.07.jpg?itok=GCsDueTC Which molecules/structures do I want to preserve? proteins: precipitation and crosslinking nucleic acids: precipitation and crosslinking polysaccharides: indirect fixation through crosslinking of proteins lipids: often extracted small molecules: go live! 3D structure: chemical fixation, freezing and chemical fixation ….there are four general fixation methods: drying heating (55°C) freezing chemical fixation www.imb‐mainz.de Microscopy Core Facility Chemical fixation (i): coagulating fixatives alcohol (methanol, ethanol, 99%) acetone Hydration state of salt (ammonium sulfate) molecules are changed » fast penetration » the antigen recognition sites for immunolabeling are preserved very well + » works nice for microtubules » the specimen shrinks almost 50% » morphology is poorly preserved ‐ » extraction of lipids/membrane proteins » incompatible with phalloidin staining for actin fibers www.imb‐mainz.de Microscopy Core Facility Chemical fixation (ii): crosslinking fixatives Aldehydes Formaldehyde Glutaraldehyde Ethylene Glycol bis‐Succinimidyl Succinate (EGS) (reversible; the crosslinks are cleaved at a pH 8.5. It is useful for membrane‐bound proteins but it is not good soluble in water) Covalent crosslinks that are determined by the active groups in each compound. www.imb‐mainz.de Microscopy Core Facility Formaldehyde/Formalin/Paraformaldehyde formaldehyde methylene hydrate paraformaldehyde (gas) (solved in water) (solid) Mw 30 g/mol (formalin) Most widely used fixative in histology good tissue penetration (~1mm/h) penetration‐fixation paradox www.imb‐mainz.de Microscopy Core Facility Formaldehyde/Formalin/Paraformaldehyde formaldehyde methylene hydrate paraformaldehyde (gas) (solved in water) (solid) Mw 30 g/mol (formalin) Formalin (100%) Paraformaldehyde form 40% (v/v) or 37% (w/v) insoluble white powder formaldehyde in water contains stabilizer (e.g. 10% methanol) degree of n = 2‐8n = up to 100 polymerization depolymerization directly when diluted at pH 7.4 pH 7.2‐7.6 and heating (65°C) www.imb‐mainz.de Microscopy Core Facility Formaldehyde fixation Formaldehyde solution needs to be fresh! Prepare from Paraformaldehyde: Paraformaldehyd (4%) • Mix 40 g PFA (Sigma) with 500 mL ultrapure water • Add 10 mL 1 M NaOH and heat to 65°C (magnetic stirrer under fume hood) • Add 100 mL 10xPBS and cool down to room temperature • Adjust pH 7.4 (1M HCL ; 1mL); • Fill to 1000 mL with ultrapure water • Sterile filtration (0.45 µm Filter) • Store aliquots at ‐20°C (ca. 6 month) Quenching solution 50 mM Tris‐Cl (pH 8.0) 100 mM NaCl www.imb‐mainz.de Microscopy Core Facility Formaldehyde fixation reaction • Formaldehyde crosslinks proteins by forming methylene bridges (‐CH2‐) between reactive groups (protein‐ protein or protein‐nucleic acid (C, A, G, no T) • Does not crosslink lipids. • Labile structures such as microtubules might not be well preserved. • Fixation is partially reversible: heat induced antigen retrieval (HIER). Kiernan JA. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and what they do. Microscopy Today. 2000;1:8–12. www.imb‐mainz.de Microscopy Core Facility Glutaraldehyde Mw 100.12 g/mol Protein cross‐linking with glutaraldehyde monomer • Introduced 1962 as EM staining (Sabatini et al. ) Protein cross‐linking with glutaraldehyde polymer • Bifunctional crosslinker small enough to penetrate tissue/ slower than formaldehyde) • Present in aqueous solutions as monomers and polymers of variable size www.imb‐mainz.de Microscopy Core Facility Fixation causes artifacts Neutral buffered formalin fixation 95% ethanol fixation A paraffin section from the mucosa of small intestine imaged at same magnification. Shrinkage (sometimes also swelling) Diffusion of unfixed material (streaming) Some artifacts can be Improper fixation avoided, some Solubilization of hydrophobic cell components Loss of protein tertiary/secondary structure reverted, some can be Antigen masking compensated… DNA/RNA: low read length, bad probe binding www.imb‐mainz.de Microscopy Core Facility Some important points… No standards exist! Fixation in neutral buffered formalin(NBF, 10%) Recommentations from the College of American Pathologists for the fixation of 8 h 3 h breast tissue in NBF ER negative ER positive sample 3‐4 mm minimum fixation time of 6‐8 h maximum fixation time 48h (24h better to prevent overfixation) fixation volume 15‐20 times higher than Estrogen receptor (ER) staining of breast carcinoma. Estrofgagrg http://www.leicabiosystems.com/pathologyleaders/effects-of-fixation-and- the bulk of the tissue tissue-processing-on-immunocytochemistry/ Effects of Fixation and Tissue Processing on Immunocytochemistry, Peter NBF solution less than 1 month old Jackson Goldstein NS, Ferkowicz MT, Odish E, Mani A, Hastah F. Minimum formalin fixation storage of fixed samples in 70% ethanol time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol 120:86-93 2003. until processing www.imb‐mainz.de Microscopy Core Facility Different kinds and combinations of fixatives www.imb‐mainz.de W.J. Howat, B.A. Wilson / Methods 70 (2014) 12–19 Microscopy Core Facility Fixatives: Alternatives to NBF www.imb‐mainz.de W.J. Howat, B.A. Wilson / Methods 70 (2014) 12–19 Microscopy Core Facility Guidelines for choosing a fixative www.imb‐mainz.de http://docs.abcam.com/pdf/kits/immunohistochemistry‐ihc‐application‐guide.pdf Microscopy Core Facility Freezing: SNAP freezing tissue blocks (max. 1cm) or organs could be snap frozen in Liquid N2 (‐196°C), use vapor phase to prevent freeze damage Isopentane on dry ice bath (‐80°C) Isopentane on liquid nitrogen (ca. ‐150 to ‐160°C) remove residual isopentane with a paper place the sample in an ice‐cold cryo‐vial storage ‐80°C or liquid nitrogen